Primer, probe, kit and detection method for detecting general H1 and H3 subtype swine influenza viruses through triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction)

Abstract

The invention discloses a primer, a probe, a kit and a detection method for detecting general H1 and H3 subtype swine influenza viruses through triple fluorescent RT-PCR (Reverse Transcription-Polymerase Chain Reaction). The detection method comprises the following steps: (1) obtaining a target sample; extracting virus RNA from the target sample; taking the extracted virus RNA as a template, adding a one-step RT-qPCR premixed solution, carrying out reverse transcription at first, and then carrying out PCR amplification; and judging a result. The establishment of the detection method can greatly shorten the epidemic disease diagnosis time, is beneficial to implementation of the principle of early discovery and early treatment in the epidemic disease treatment process, provides an operation basis for animal epidemic disease diagnosis and flow regulation monitoring in China, and has very important significance for promoting standardization of animal epidemic disease diagnosis in China.

Description

Technical Field

[0001] The present invention relates to the field of biological detection, and in particular to primers, probes, kits and detection methods for detecting universal, H1 and H3 subtype swine influenza viruses by triple fluorescence RT-PCR. Background Art

[0002] Swine influenza (SI) is an acute, febrile, highly contagious respiratory disease of pigs caused by swine influenza viruses (SIVs). Clinically, it is characterized by sudden onset, high fever, cough, dyspnea, repeated attacks, and high morbidity. Pigs are common susceptible hosts for influenza viruses from birds, pigs, and humans. They are "mixers" for influenza virus gene recombination or reassortment and "incubators" for the production of new epidemic strains of influenza viruses. Newly produced epidemic strains are likely to break through species restrictions and cause zoonotic transmission events. The number of cases of cross-species infection of new three-source (human, bird, and pig) swine influenza viruses is increasing, seriously threatening public health security. In swine influenza, in addition to the H1 and H3 subtypes, other subtypes of influenza A viruses are prevalent in pig populations, such as the H5N1 subtype swine influenza virus. Studies have suggested that the avian-like H1N1 subtype swine influenza virus is the virus with the greatest possibility of causing a human influenza pandemic and should be given high attention.

[0003] The identification of swine influenza virus subtype infection based on epidemiological surveys, clinical symptoms, pathological anatomical changes and other methods is time-consuming and has low accuracy. Since its birth, fluorescent quantitative RT-PCR, as a new type of molecular biology technology, has become one of the important means of invention research due to its high specificity and sensitivity, and can obtain a large number of target fragments in a short time. At present, there is no real-time triple fluorescent quantitative RT-PCR detection kit and method for swine influenza virus that can simultaneously distinguish and detect the universal type, H1 subtype, and H3 subtype of influenza A virus in China. Summary of the invention

[0004] The purpose of the present invention is to solve the shortcomings of the prior art, and to propose primers, probes, kits and detection methods for detecting universal swine influenza virus, H1 and H3 subtype swine influenza virus by triple fluorescence RT-PCR. The primers, probes and detection methods of the present application can quickly identify the subtype of swine influenza virus infection, and can also detect other type A influenza virus infections, which is beneficial to the inhibition and prevention of swine influenza virus infection and transmission, and reduces economic losses. The entire detection process is simple, the detection time is short, and the sensitivity is significantly improved.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The invention discloses primers for detecting universal type, H1 and H3 subtype swine influenza viruses of swine influenza viruses by triple fluorescence RT-PCR, universal forward and reverse primers for detecting swine influenza viruses, forward and reverse primers for detecting H1 subtype swine influenza viruses, and forward and reverse primers for detecting H3 subtype swine influenza viruses. The universal primer sequence of swine influenza viruses is derived from the M gene of type A influenza viruses, and the M gene sequences of different subtypes of type A influenza viruses are obtained from the GenBank database for comparison. The forward and reverse primer sequences for detecting type A influenza viruses are shown in SEQ ID NOs: 001 and 002 based on the conservative sequence of the M gene. The forward and reverse primer sequences for detecting H1 subtype swine influenza viruses are derived from the HA gene of the H1 subtype swine influenza viruses, and the HA gene sequences of the H1 subtype swine influenza viruses are obtained from the GenBank database for comparison. The forward and reverse primer sequences for detecting H1 subtype swine influenza viruses are shown in SEQ ID NOs: 004 and 005 based on the conservative sequence of the M gene. NO: 005; the forward and reverse primers for detecting H3 subtype swine influenza virus are derived from the HA gene of H3 subtype swine influenza virus. The HA gene sequence of H3 subtype swine influenza virus is obtained from the GenBank database for comparison, and is designed based on its conservative sequence. The forward and reverse primer sequences for detecting H3 subtype swine influenza virus are shown in SEQ ID NO: 007 and SEQ ID NO: 008; the detection primers are compared by Primer-BLAST in NCBI to ensure the specificity of the designed primers.

[0007] The present invention also discloses the triple fluorescence RT-PCR probe for detecting universal swine influenza virus, H1 and H3 subtype swine influenza virus. The primer sequence of the amplification probe for detecting universal swine influenza virus is shown in SEQ ID NO: 003; the primer sequence of the amplification probe for detecting H1 subtype swine influenza virus is shown in SEQ ID NO: 006; and the primer sequence of the amplification probe for detecting H3 subtype swine influenza virus is shown in SEQ ID NO: 009.

[0008] The present invention also discloses a kit for detecting swine influenza virus, which comprises at least one of the above-mentioned detection primers and at least one of the above-mentioned primer probes.

[0009] Preferably, the kit further comprises: an RNA extraction reagent, a one-step RT-qPCR reaction reagent, a negative control and a positive control.

[0010] The present invention also discloses a method for detecting universal, H1, and H3 subtype swine influenza viruses by triple fluorescence RT-PCR, comprising the following steps:

[0011] (1) Obtaining target samples;

[0012] (2) extracting viral RNA from the target sample;

[0013] (3) Using the extracted viral RNA as a template, add RT-qPCR premix solution, perform reverse transcription first, and then perform fluorescent PCR amplification;

[0014] (4) Result determination;

[0015] Preferably, the RT-qPCR premix described in step (3) includes forward and reverse primers for detecting universal swine influenza virus, the primer sequences are shown in SEQ ID NO: 001 and SEQ ID NO: 002, and the amplification probe primer sequence is shown in SEQ ID NO: 003; forward and reverse primers for detecting H1 subtype swine influenza virus, the primer sequences are shown in SEQ ID NO: 004 and SEQ ID NO: 005, and the amplification probe primer sequence is shown in SEQ ID NO: 006; forward and reverse primers for detecting H3 subtype swine influenza virus, the primer sequences are shown in SEQ ID NO: 007 and SEQ ID NO: 008, and the amplification probe primer sequence is shown in SEQ ID NO: 009.

[0016] Preferably, in any of the above schemes, the PCR amplification reaction conditions in step (4) are: 55°C, 15 min; pre-denaturation at 95°C, 3 min; 95°C, 15 s, 54°C, 30 s and collecting fluorescence signals, for a total of 45 cycles.

[0017] In any of the above solutions, preferably, the condition for determining the result in step (4) is:

[0018] (a) Negative control FAM, VIC, and Cy5 channels have no CT value or a CT value greater than 38, and no specific amplification curve;

[0019] (b) The CT values ​​of the positive control FAM, VIC, and Cy5 channels were less than 30, and all had specific amplification curves.

[0020] In any of the above schemes, preferably, the method for determining the result in step (4) is:

[0021] (1) When there is no Ct value in the FAM channel, VIC channel, and Cy5 channel or the Ct values ​​are all greater than 38 and there is no specific amplification curve, the sample is negative for swine influenza virus nucleic acid;

[0022] (2) When the Ct value of the FAM channel is less than or equal to 35 and there is a specific amplification curve, and the VIC channel and Cy5 channel have no Ct value or the Ct value is greater than 38 and there is no specific amplification curve, the sample is positive for universal influenza A virus nucleic acid;

[0023] (3) When the Ct values ​​of the FAM channel and the VIC channel are less than or equal to 35 and there is a specific amplification curve, and the Cy5 channel has no Ct value or the Ct values ​​are greater than 38 and there is no specific amplification curve, the sample is positive for swine influenza H3 subtype virus nucleic acid;

[0024] (4) When the Ct values ​​of the FAM channel and the Cy5 channel are less than or equal to 35 and there is a specific amplification curve, and the VIC channel has no Ct value or the Ct values ​​are greater than 38 and there is no specific amplification curve, the sample is positive for swine influenza H1 subtype virus nucleic acid;

[0025] (5) When the Ct values ​​of the FAM channel, Cy5 channel, and VIC channel are all less than or equal to 35, and there is a specific amplification curve, the sample is positive for swine influenza H1 and H3 subtype virus nucleic acid;

[0026] (6) If the Ct values ​​of the FAM channel, VIC channel, and Cy5 channel are between 35 and 38 and there is a specific amplification curve, the sample is suspicious and needs to be retested.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] In view of the prevalence of other subtypes of universal influenza A virus in pigs (such as H5N1 subtype swine influenza virus, etc.) in addition to H1 subtype and H3 subtype in swine influenza, the present invention establishes a real-time triple fluorescence quantitative RT-PCR method for swine influenza virus that can simultaneously detect universal influenza A virus, H1 subtype, and H3 subtype. The application of this method can, on the one hand, type the subtypes of swine influenza infected in pigs, and at the same time detect the infection of other subtypes of swine influenza. On the other hand, it can greatly shorten the time for disease diagnosis, help implement the principle of "early detection and early treatment" in the process of disease disposal, provide an operational basis for the diagnosis and monitoring of animal diseases in my country, and have a very important significance for promoting the standardization of animal disease diagnosis in my country.

[0029] The invention designs specific primers for the influenza A virus, H1 subtype and H3 subtype swine influenza virus currently prevalent in my country, and has the characteristic of strong specificity.

[0030] The invention has a wide range of applications and is suitable for detecting swine influenza virus nucleic acid in samples such as pig tissues, nasopharyngeal swabs, pig feces, chicken embryo cultures, cell culture supernatants, and environmental samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the ATY standard curve;

[0032] Figure 2 is the H1 standard curve;

[0033] Figure 3 is the H3 standard curve;

[0034] Figure 4 The ATY sensitivity results are as follows: 1.2×10 7 ~1.2×10 0 copies / μL;

[0035] Figure 5 H1 sensitivity results, from left to right are 1.2×10 7 ~1.2×10 0 copies / μL;

[0036] Figure 6 H3 sensitivity results, from left to right are 1.2×10 7 ~1.2×10 0 copies / μL;

[0037] Figure 7 The specificity test results of Example 1 are shown in FIG. DETAILED DESCRIPTION

[0038] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention.

[0039] The triple fluorescence RT-PCR detection method of the present invention for detecting universal influenza A virus, H1 and H3 subtype swine influenza virus has the following specific application scope and implementation steps:

[0040] 1. Scope of application

[0041] It is suitable for detecting swine influenza virus nucleic acid in samples such as pig tissues, nasopharyngeal swabs, chicken embryo cultures, cell culture supernatants, and environmental samples.

[0042] 2. Equipment

[0043] Fluorescence quantitative PCR instrument, Class II biological safety cabinet, desktop high-speed refrigerated centrifuge, UV spectrophotometer, micropipette, centrifuge tube, 8-row fluorescent PCR tube or 96-well fluorescent PCR plate, sealing film, gun tip, etc.

[0044] 3. Materials and reagents

[0045] Positive samples or viruses such as PRRSV, PRV, PCV2, PCV3, PEDV, TGEV, CSFV, etc., avian influenza virus antigens such as H5, H7, H9, swine influenza virus antigens such as H1, H3, One-step RT-qPCR reaction solution, plasmid extraction kit.

[0046] 4. Primer design and synthesis

[0047] Obtain the HA and M genes of H1 and H3 subtype swine influenza viruses and other subtype influenza viruses from GenBank. Use MEGA software to compare and analyze the gene sequences of the obtained H1 and H3 subtype swine influenza virus HA genes and influenza virus M genes, and screen the conservative gene sequences of each gene. Use biological software to design specific primers and probes, and use Primer-BLAST in NCBI to compare and ensure the specificity of the designed primers.

[0048] 5. Sample processing

[0049] The sampling tube containing the nasal or throat swab is first shaken with a vortex mixer for 5 to 10 seconds, and then the head of the cotton swab is repeatedly squeezed against the bottle wall with sterile tweezers and taken out. It is centrifuged at 4°C and 3000r / min for 10min, and the supernatant is taken for later use.

[0050] Use sterile scissors and forceps to cut soybean-sized tissue blocks from trachea, lung and other pathological samples, add 1 mL of pH 7.2 phosphate buffer, grind thoroughly, centrifuge at 5000g / min for 3 minutes, and take the supernatant for later use.

[0051] 6. Total RNA Extraction

[0052] Total RNA was extracted from the processed cotton swab supernatant and tissue homogenate supernatant according to the instructions of the extraction reagent provided in the kit.

[0053] 7. Preparation of Standards

[0054] The HA gene and M gene of H1 and H3 subtype swine influenza virus were synthesized into gene fragments and connected to the PUC57 vector, transformed, and a single colony was picked, shaken and plasmids were extracted. The recombinant plasmids with correct sequencing were used as standards. The concentration of the recombinant plasmids was measured by UV spectrophotometer and the copy number was calculated.

[0055] 8. Optimization of triple fluorescence quantitative PCR reaction system

[0056] Using standard plasmid as template, the reaction annealing temperature and amplification method (2-step method and 3-step method) were optimized, and then the primer and probe concentrations were optimized using the matrix method. Finally, the reaction system (20μL, 25μL, 30μL, 35μL, 40μL, 45μL) was optimized to obtain the optimal reaction conditions and system for triple fluorescence quantitative RT-PCR.

[0057] 9. Sensitivity test and establishment of standard curve

[0058] The standard plasmid was diluted 10-fold in 7 steps. 7 ~10 0The optimized triple fluorescence quantitative RT-PCR reaction system was used to amplify the templates with 8 orders of magnitude of copy number to determine the minimum detection limit. The logarithm of template concentration was used as the X-axis and the Ct value was used as the Y-axis. The standard curve was drawn using Graphpad and linear regression analysis was performed.

[0059] 10. Specificity test

[0060] Total RNA was extracted from viruses or positive samples such as PRV, CSFV, PRRSV, PCV2, PCV3, PEDV, TGEV, SVA, influenza virus antigens such as H5, H7, H9, and swine influenza virus antigens of H1 and H3 subtypes. PCR-grade sterile enzyme-free water was used as a negative control. The results were tested according to the optimized reaction conditions and reaction system to evaluate the specificity of the method.

[0061] 11. Repeatability test

[0062] Take 10 3 , 10 4 , 10 5 The copy standard plasmid was used as a template to optimize the reaction conditions and system to carry out intra-batch and inter-batch repeated experiments, and the mean, standard deviation and coefficient of variation within and between batches were calculated to evaluate the repeatability of the method.

[0063] Example 1

[0064] 1.1 Materials and reagents

[0065] Viruses or positive samples such as PRV, CSFV, PRRSV, PCV2, PCV3, PEDV, TGEV, and RV were stored in the laboratory of the applicant unit; influenza virus antigens such as H5, H7, and H9, and swine influenza virus antigens such as H1 and H3 were purchased from Guosheng Biotechnology Co., Ltd., and One-step RT-qPCR reaction solution was purchased from Novozyme Biotech Co., Ltd.; plasmid extraction kits were purchased from OMEGA; and competent cells were purchased from Kangwei Century Biotechnology Co., Ltd.

[0066] 1.2 Primer and probe design

[0067] According to the gene sequences of HA and M genes of H1 and H3 subtype swine influenza viruses and other subtype influenza viruses included in GenBank, MEGA software was used for comparison analysis to screen the conservative sequence regions for designing primers and probes, and then PrimerPremier 5.0 software was used to design specific primers and probes. The primer and probe sequences were verified by Primer-BLAST for primer and probe specificity. The primer and probe sequences are shown in Table 1 and were synthesized by Boshang Biotechnology Co., Ltd.

[0068] Table 1 Primers and probes for RT-qPCR of swine influenza virus

[0069]

[0070] 1.3 Preparation of Standards

[0071] The target gene fragments such as the H1 subtype swine influenza virus HA gene, the H3 subtype swine influenza virus HA gene and the A type influenza virus M gene were connected in series and synthesized, connected to the PUC57 vector, transformed, picked a single colony, shaken and extracted the plasmid, and the recombinant plasmid with the correct sequencing was used as the standard. The concentration was determined and the standard plasmid concentration was calculated to be 4.56×10 10 copies / μL.

[0072] 1.4 Optimization of reaction conditions and reaction system

[0073] Fluorescence quantitative PCR was performed using the standard as a template, and the annealing temperature and amplification method (2-step method and 3-step method) were optimized. Subsequently, under the optimal reaction conditions, the amount of primers and probes was optimized using the matrix method. Under the optimized reaction conditions and the amount of primers and probes, the reaction system was optimized.

[0074] 1.5 Sensitivity test and standard curve

[0075] The standard plasmid was diluted 10-fold in 7 steps. 7 ~10 0 The optimized triple fluorescence quantitative RT-PCR reaction system was used to amplify the templates with 8 orders of magnitude of copy number to determine the minimum detection limit. The logarithm of template concentration was used as the X-axis and the Ct value was used as the Y-axis. The standard curve was drawn using Graphpad and linear regression analysis was performed.

[0076] 1.6 Specificity test

[0077] Total RNA was extracted from viruses or positive samples such as PRV, CSFV, PRRSV, PCV2, PCV3, PEDV, TGEV, SVA, influenza virus antigens such as H5, H7, H9, and swine influenza virus antigens of H1 and H3 subtypes. PCR-grade sterile enzyme-free water was used as a negative control. The results were tested according to the optimized reaction conditions and reaction system to evaluate the specificity of the method.

[0078] 1.7 Repeatability test

[0079] Take 10 3 , 10 4 , 10 5 The copy standard plasmid was used as a template to optimize the reaction conditions and system to carry out intra-batch and inter-batch repeated experiments, and the mean, standard deviation and coefficient of variation within and between batches were calculated to evaluate the repeatability of the method.

[0080] According to the above method, the following results were obtained: the optimal reaction conditions were determined to be 55°C, 15min; 95°C for 3min; 95°C for 15s, 54°C for 30s (fluorescence absorption), 45 cycles. The optimal amounts of primers for universal influenza A virus, H1 subtype, and H3 subtype swine influenza virus were 0.6μL, 0.8μL, and 0.6μL, respectively, and the optimal amounts of probes were 0.4μL, 0.6μL, and 0.4μL, respectively; the optimal reaction system was 40μL, and the components of the reaction system were shown in Table 2.

[0081] Table 2 Optimal reaction system

[0082]

[0083] Take 10 0 ~10 7 The standard plasmid was used as a template and detected by fluorescence quantitative PCR. 2 ~10 7 The linear relationship was good within the concentration range of copies / μL. The standard curve was drawn with the logarithm of the standard plasmid concentration as the X-axis and the C t value as the Y-axis, and a linear regression analysis was performed to obtain the universal fluorescence quantitative PCR detection standard curve for influenza A virus (ATY): Y = -4.082*X + 48.01 ( Figure 1 ), the standard curve of fluorescence quantitative PCR detection of H1 subtype swine influenza virus was obtained as Y=-3.770*X+42.40( Figure 2 ), the standard curve of fluorescence quantitative PCR detection of H3 subtype swine influenza virus was obtained as Y=-4.155*X+46.11( Figure 3 ). The toxic content of influenza A virus and H1 and H3 subtype swine influenza virus in the sample to be tested can be calculated by referring to the standard curve.

[0084] The results of the sensitivity test showed that the detection limit of influenza A universal strain in the triple fluorescence quantitative PCR detection method was 1.2×10 2 copies / μL( Figure 4 ), the detection limit of H1 subtype swine influenza virus is 1.2×10 1 copies / μL( Figure 5 ), the detection limit of H3 subtype swine influenza virus is 1.2×10 2 copies / μL( Figure 6 ).

[0085] like Figure 4-6 As shown, the concentration of standard plasmids from left to right is 1.2×10 7 ~1.2×10 0 copies / μL;

[0086] Using standard plasmid as positive control, PRV, CSFV, PRRSV, PCV2, PCV3, PEDV, TGEV, rotavirus and other viruses or positive samples, H5, H7, H9 and other subtype influenza virus antigens and H1, H3 subtype swine influenza virus antigens and other samples as samples, extracting nucleic acid (DNA / RNA), using them as templates, and using triple fluorescence quantitative PCR detection method to detect different samples. The results showed that the samples of H5, H7, H9 and other subtype influenza virus antigens had specific amplification curves in FAM channel detection, the samples of H1 subtype swine influenza virus antigen had specific amplification curves in FAM and Cy5 channel detection, the samples of H3 subtype swine influenza virus antigen had specific amplification curves in FAM and VIC channel detection, the positive control samples had specific amplification curves in FAM, Cy5 and VIC channel detection, and other samples and negative controls had no specific amplification curves, indicating that this method has good specificity. The specificity experimental results are as follows: Figure 7 As shown, specific results (blue FAM channel: ATY; red CY5 channel: H1; green VIC channel: H3)

[0087] The results of the repeatability test showed that the coefficient of variation of the intra-batch repeatability test for universal swine influenza virus detection was 1.30% to 2.95%, and the coefficient of variation of the inter-batch repeatability test was 0.90% to 4.92% (Table 3); the coefficient of variation of the intra-batch repeatability test for H1 subtype swine influenza virus detection was 0.45% to 0.47%, and the coefficient of variation of the inter-batch repeatability test was 0.37% to 0.87% (Table 4); the coefficient of variation of the intra-batch repeatability test for H3 subtype swine influenza virus detection was 0.45% to 1.55%, and the coefficient of variation of the inter-batch repeatability test was 0.37% to 0.87% (Table 5); the above repeatability test results indicate that the triple fluorescence quantitative PCR method established by the present invention has good repeatability and stability.

[0088] Table 3 General-FAM channel test results

[0089]

[0090] Table 4H1-CY5 channel test results

[0091]

[0092] Table 5H3-VIC channel test results

[0093]

[0094] The above are only preferred specific implementation modes of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical solutions and inventive concepts of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Primers for triple fluorescent RT-PCR detection of universal, H1, and H3 subtype swine influenza viruses, characterized in that: The detection primers include universal forward and reverse primers for detecting swine influenza, forward and reverse primers for H1 subtype swine influenza virus, and forward and reverse primers for H3 subtype swine influenza virus. The universal swine influenza primer sequence is derived from the M gene of type A influenza virus. The M gene sequences of different subtypes of type A influenza virus are obtained from the GenBank database for comparison and are designed based on the conservative sequence of the M gene. The forward and reverse primer sequences for detecting universal swine influenza are shown in SEQ ID NO: 001 and SEQ ID NO:

002. The forward and reverse primers for detecting H1 subtype swine influenza virus are derived from the HA gene of H1 subtype swine influenza virus. The HA gene sequence of H1 subtype swine influenza virus is obtained from the GenBank database for comparison and is designed based on its conservative sequence. The forward and reverse primer sequences for detecting H1 subtype swine influenza virus are shown in SEQ ID NO: 004 and SEQ ID NO:

005. NO: 005; the forward and reverse primers for detecting H3 subtype swine influenza virus are derived from the HA gene of H3 subtype swine influenza virus. The HA gene sequence of H3 subtype swine influenza virus is obtained from the GenBank database for comparison and designed based on its conservative sequence. The forward and reverse primer sequences for detecting H3 subtype swine influenza virus are shown in SEQ ID NO: 007 and SEQ ID NO:

008.

2. A triple fluorescent RT-PCR probe for detecting universal, H1, and H3 subtype swine influenza viruses, characterized in that: The universal amplification probe primer sequence for detecting swine influenza virus is shown in SEQ ID NO: 003; the amplification probe primer sequence for detecting H1 subtype swine influenza virus is shown in SEQ ID NO: 006; and the amplification probe primer sequence for detecting H3 subtype swine influenza virus is shown in SEQ ID NO:

009.

3. A kit for detecting swine influenza virus, characterized in that: The kit comprises at least one detection primer according to claim 1 and at least one primer probe according to claim 2.

4. The kit for detecting swine influenza virus according to claim 3 is a kit for detecting universal swine influenza virus, H1, and H3 subtype swine influenza virus, characterized in that: The kit also includes: RNA extraction reagent, one-step RT-qPCR reaction reagent, negative control and positive control.

5. A method for detecting universal, H1, and H3 subtype swine influenza viruses by triple fluorescence RT-PCR, characterized in that: The following steps are involved: (1) Obtain target samples; (2) Extracting viral RNA from target samples; (3) Using the extracted viral RNA as a template, add the one-step RT-qPCR premix, perform reverse transcription first, and then perform fluorescent PCR amplification; (4) Result determination 6. The method for detecting universal, H1 and H3 subtype swine influenza viruses by triple fluorescence RT-PCR according to claim 5, characterized in that: The one-step RT-qPCR premix in step (3) includes forward and reverse primers for detecting universal influenza A virus, the primer sequences are shown in SEQ ID NO: 001 and SEQ ID NO: 002, and the amplification probe primer sequence is shown in SEQ ID NO: 003; forward and reverse primers for detecting H1 subtype swine influenza virus, the primer sequences are shown in SEQ ID NO: 004 and SEQ ID NO: 005, and the amplification probe primer sequence is shown in SEQ ID NO: 006; forward and reverse primers for detecting H3 subtype swine influenza virus, the primer sequences are shown in SEQ ID NO: 007 and SEQ ID NO: 008, and the amplification probe primer sequence is shown in SEQ ID NO:

009.

7. The method for detecting universal, H1 and H3 subtype swine influenza viruses by triple fluorescence RT-PCR according to claim 5, characterized in that: The PCR amplification reaction conditions in step (4) were as follows: 55°C, 15 min; pre-denaturation at 95°C, 3 min; 95°C, 15 s, 54°C, 30 s and collecting fluorescence signals, for a total of 45 cycles.

8. The method for detecting universal, H1 and H3 subtype swine influenza viruses by triple fluorescence RT-PCR according to claim 5, characterized in that: The conditions for determining the result in step (4) are: (a) The negative control FAM, VIC, and Cy5 channels have no CT value or a CT value greater than 38, and no specific amplification curve; (b) The CT values ​​of the positive control FAM, VIC, and Cy5 channels were less than 30, and all had specific amplification curves.

9. The method for detecting universal, H1 and H3 subtype swine influenza viruses by triple fluorescence RT-PCR according to claim 5, characterized in that: The method for determining the result in step (4) is: (1) When there is no Ct value in the FAM channel, VIC channel, and Cy5 channel or the Ct values ​​are all greater than 38 and there is no specific amplification curve, the sample is negative for swine influenza virus nucleic acid; (2) When the Ct value of the FAM channel is less than or equal to 35 and there is a specific amplification curve, and the VIC channel and Cy5 channel have no Ct value or the Ct values ​​are greater than 38 and there is no specific amplification curve, the sample is positive for universal influenza A virus nucleic acid; (3) When the Ct values ​​of the FAM channel and the VIC channel are less than or equal to 35 and there is a specific amplification curve, and the Cy5 channel has no Ct value or the Ct values ​​are greater than 38 and there is no specific amplification curve, the sample is positive for swine influenza H3 subtype virus nucleic acid; (4) When the Ct values ​​of the FAM channel and the Cy5 channel are less than or equal to 35 and there is a specific amplification curve, and the VIC channel has no Ct value or the Ct values ​​are greater than 38 and there is no specific amplification curve, the sample is positive for swine influenza H1 subtype virus nucleic acid; (5) When the Ct values ​​of the FAM channel, Cy5 channel, and VIC channel are all less than or equal to 35, and there is a specific amplification curve, the sample is positive for swine influenza H1 and H3 subtype virus nucleic acid; (6) If the Ct values ​​of the FAM channel, VIC channel, and Cy5 channel are between 35 and 38 and there is a specific amplification curve, the sample is suspicious and needs to be retested.

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